The loss of CD28 expression is the most consistent marker of replicative senescence in T cells, and is a predictor of age-associated immunoincompetence in humans. CD28null T cells are highly oligoclonal with shortened telomeres indicating their long replicative history. Since CD28 is essential for T cell proliferation and effector function, observations that CD28null T cells are derived from CD28+ progenitors led to the hypothesis that downregulation of CD28 expression underlies a program of T cell senescence. Molecular studies show that human CD28 gene transcription is regulated by the initiator (INR), a novel core promoter element consisting of contiguous alpha and beta motifs that function as a unit. This alphabeta-INR is inactive in CD28null T cells due to the coordinated lack of site alpha- and beta-specific transcription factors. In CD28+ T cells, proliferative signals downregulate alphabeta-1NR activity resulting in the modulation, if not loss, of CD28 indicating that the temporal silencing of alphabeta-1NR is central to the transformation of highly proliferative CD28+ T cells into senescent CD28null T cells. The identification of nucleolin and the A-isoform of heterogeneous ribonucleoprotein-D0 (hnRNP-D0A) as components of the alpha-bound transcription factor complex will permit the molecular dissection of alphabeta-INR-dependent expression of CD28. Since these are commonly expressed proteins, studies have been designed to examine the basis of nucleolin/hnRNP-D0A complex formation in CD28+, but not CD28null ,T cells. Because discrete sequence modules dictate the functions of these proteins, studies will be conducted to identify structural variants of nucleolin and/or hnRNP-D0A that account for a protein complex with functional specificity for the trans-activation of the alphabeta-INR. Specific studies are also designed to examine modifications of nucleolin and/or hnRNP-DoA that modulate alphabeta-INR function during CD28+-->CD28 null phenotypic transition that accompanies T cell proliferation. Complementary studies have also been proposed to evaluate the role of other putative cis-acting elements and trans-acting factors in the orchestration of CD28 transcription by the alphabeta-INR,and how they influence the development of CD28null T cells. Finally, studies have been designed to examine the molecular/biochemical basis for the opposing effects of tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 in alphabeta-1NR trans-activation. Whereas TNFalpha represses alphabeta-INRfunction, IL-12 restores it. Hence, regulation of alphabeta-INR by TNFalpha and IL-12 will help elucidate the pathway(s) that accelerate development/accumulation of senescent CD28null T cells in vivo. Understanding the intricacies of alphabeta-INR-dependent control of CD28 expression could be important for future development of strategies to delay T cell senescence and/or reconstitute classical T cell function in otherwise senescent cells.